EP2200658A2 - Positronen-emissionstomographie-sonden zur darstellung von immunaktivierung und ausgewählten krebsarten - Google Patents

Positronen-emissionstomographie-sonden zur darstellung von immunaktivierung und ausgewählten krebsarten

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Publication number
EP2200658A2
EP2200658A2 EP08831942A EP08831942A EP2200658A2 EP 2200658 A2 EP2200658 A2 EP 2200658A2 EP 08831942 A EP08831942 A EP 08831942A EP 08831942 A EP08831942 A EP 08831942A EP 2200658 A2 EP2200658 A2 EP 2200658A2
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EP
European Patent Office
Prior art keywords
fluoro
fac
deoxy
pet probe
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP08831942A
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English (en)
French (fr)
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EP2200658B1 (de
Inventor
Caius G. Radu
Owen N. Witte
Evan David Nair-Gill
Nagichettiar Satyamurthy
Chengyi J. Shu
Johannes Czernin
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University of California
University of California Berkeley
University of California San Diego UCSD
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University of California
University of California Berkeley
University of California San Diego UCSD
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis

Definitions

  • PET Positron Emission Tomography
  • a PET probe includes a compound having a structure according to Formula IA and/or Formula IB,
  • Ri can be Ff, OH, or F
  • R 2 can be H, OH, or F
  • R 3 can be H or F
  • R 6 can be H or F.
  • A can be selected from the group consisting of
  • R 4 can be H, halogen, or alkyl from 1 to 6 carbons.
  • R 5 can be H, halogen, or alkyl from 1 to 6 carbons.
  • One or more of Ri, R 2 , R 3 , R 4 , R 5 , and R 6 can be a radioisotope, for example, 18 F, 76 Br and 124 I.
  • A can be
  • Ri can be OH or fluorine
  • R 2 can be hydrogen or fluorine
  • R 3 can be fluorine
  • R 4 can be H, F, Cl, Br, I, CH 3 , or C 2 H 5 , and one or more of R
  • A can be
  • Ri can be OH or fluorine
  • R 2 can be hydrogen
  • R 3 can be fluorine
  • R 5 can be Cl, F
  • R 2 , R 3 , and R 5 can be F.
  • R 4 can be H, F, Cl, Br, or CH 3
  • R 5 can be Cl, R
  • R 2 and R 5 can be not radioisotopes other than in a naturally occurring proportion.
  • the PET probe can include one or more pharmaceutically acceptable carriers and/or excipients.
  • pharmaceutically acceptable carriers and/or excipients One of ordinary skill in the art will be able to selection appropriate pharmaceutically acceptable carriers and/or excipients based on an envisioned application.
  • the PET probe can be a dCK substrate.
  • the PET probe can be a dCK substrate.
  • PET probe can be resistant to deamination, for example, resistant to deamination by cytidine deaminase (CDA) or adenosine deaminase.
  • CDA cytidine deaminase
  • the PET probe resistant to deamination can be [ 18 F]L-FAC, [ 18 F]L-FXAC, [ 18 F]L-FBAC, [ 18 F]L- FCAC, [ 18 F]L-FFAC, [ 18 F]L-FRAC, [ 18 F]L-FMAC, [ 18 F]F-CA, D-2- 18 F-CA, L-2- 18 F-CA, D-3- 18 F-CA, and L-3- 18 F-CA.
  • the PET probe can be used in the diagnosis and treatment of a condition selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, EAE (Experimental Autoimmune Encephalomyelitis), multiple sclerosis, atherosclerosis, an autoimmune disorder, and cancer.
  • the PET probe can be used to evaluate the efficacy in the treatment of cancer of anticancer agents that are taken up into cells via nucleoside transporters and deoxycytidine kinase (dCK)-mediated phosphorylation.
  • dCK deoxycytidine kinase
  • a method of synthesizing a PET probe according to the invention can include the following.
  • 2-O-[(trifluoromethyl)sulfonyl]-l,3,5-tri-O-benzoyl- ⁇ -D- ribofuranose an isomer of 5-benzoyloxymethyl-4,2-benzoyloxy-3- trifluoromethylsulfonatofuran
  • [ 18 F]fluoride ion can be reacted with [ 18 F]fluoride ion.
  • 2-deoxy-2-[ F]fluoro-3,5-di-O-benzoyl- ⁇ -D-arabinofuranosyl bromide an isomer of 5-benzoyloxymethyl-4-benzoyloxy-3- 18 fluoro-2-bromofuran
  • 4-N-(trimethylsilyl)-2-O-(trimethylsilyl)pyrimidine-4-arnine i.e., N- (trimethylsilyl)-2-((trimethylsilyl)oxy)pyrimidin-4-amine
  • l-(2'-deoxy-2'- [ l8 F]fluoro-3,5-di-O-benzoyl- ⁇ -D-arabinofuranosyl)cytosine (an isomer of l-(4- benzoyloxymethyl-3-benzoyloxy-2-deoxy-2- 18 fluoroarabinofuranosyl)cytosine) can be reacted with an alkoxide to form the PET probe.
  • the alkoxide can be, for example, an alkalki methoxide, e.g., sodium methoxide.
  • a method of synthesizing a PET probe according to the invention can include the following.
  • 2-O-[(Trifluoromethyl)sulfonyl]-l,3,5-tri-O-benzoyl- ⁇ -L- ribofuranose can be reacted with [ 18 F] fluoride ion to form 2-deoxy-2-[ 18 F]fluoro- 1,3,5-tri-O-benzoyl- ⁇ -L-arabinofuranose as a first radiolabeled intermediate.
  • the first radiolabeled intermediate can be reacted with hydrogen bromide to form 2- deoxy-2-[ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -L-arabinofuranosyl bromide as a second radiolabeled intermediate.
  • the second radiolabeled intermediate can be reacted with 4-N-(trimethylsilyl)-2-O-(trimethylsilyl)pyrimidine-4-amine to form l-(2'-deoxy-2'- [ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -L-arabinofuranosyl)cytosine as a third radiolabeled intermediate.
  • the third radiolabeled intermediate can be reacted with an alkoxide to form the [ 18 F]L-FAC PET probe.
  • a method of synthesizing an [ 18 F]-CA PET probe according to the invention can include the following. 2-chloroadenosine can be reacted with monomethoxytrityl chloride to form a first intermediate. The first intermediate can be reacted with trifyl chloride to form a second intermediate. And the second intermediate can be reacted with [ 18 F] fluoride ion to form the [ 18 F]-CA PET probe.
  • an [ 18 F]D-CA PET probe can be synthesized by reacting D-2- chloroadenosine and monomethoxytrityl chloride to form the first intermediate, which can be reacted with [ l 8 F]fluoride ion to form the [ 18 F]D-CA PET probe.
  • an [ 18 F]L-CA PET probe can be synthesized by reacting L-2-chloroadenosine and monomethoxytrityl chloride to form the first intermediate, which can be reacted with [ 18 F]fluoride ion to form the [ 18 F]L-CA PET probe..
  • a method of synthesizing an [ 18 F]D-FXAC PET probe according to the present invention can include the following. 2-O-[(Trifluoromethyl)sulfonyl]- 1,3,5- tri-O-benzoyl- ⁇ -D-ribofuranose can be reacted with [ 18 F] fluoride ion to form 2- deoxy-2-[ F]fiuoro-l ,3,5-tri-0-benzoyl-oc-D-arabinofuranose as a first radiolabeled intermediate.
  • the first radiolabeled intermediate can be reacted with hydrogen bromide to form 2-deoxy-2-[ l 8 F]fluoro-3,5-di-0-benzoyl- ⁇ -D-arabinofuranosyl bromide as a second radiolabeled intermediate.
  • the second radiolabeled intermediate can be reacted with 5-halo-4-N-(trimethylsilyl)-2-O-(trimethylsilyl)pyrimidine-4- amine to form 5-halo-l-(2'-deoxy-2'-[ l 8 F]fluoro-3,5-di-O-benzoyl- ⁇ -D- arabinofuranosyl)cytosine as a third radiolabeled intermediate.
  • halo can be fluoro, chloro, or bromo.
  • a method of synthesizing an [ 18 F]L-FXAC PET probe according to the present invention can include the following.
  • 2-O-[(Trifluoromethyl)sulfonyl]-l,3,5- tri-O-benzoyl- ⁇ -L-ribofuranose can be reacted with [ l 8 F]fluoride ion to form 2- deoxy-2-[ l 8 F]fluoro-l,3,5-tri-O-benzoyl- ⁇ -L-arabinofuranose as a first radiolabeled intermediate.
  • the first radiolabeled intermediate can be reacted with hydrogen bromide to form 2-deoxy-2-[ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -L-arabinofuranosyl bromide as a second radiolabeled intermediate.
  • the second radiolabeled intermediate can be reacted with 5-halo-4-N-(trimethylsilyl)-2-O-(trimethylsilyl)pyrimidine-4- amine to form 5-halo-l-(2'-deoxy-2'-[ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -L- arabinofuranosyl)cytosine as a third radiolabeled intermediate.
  • a method of synthesizing an [ 18 F]D-FRAC PET probe according to the present invention can include the following.
  • 2-O-[(Trifluoromethyl)sulfonyl]- 1,3,5- tri-O-benzoyl- ⁇ -D-ribofuranose can be reacted with [ l 8 F]fluoride ion to form 2- deoxy-2-[ 18 F]fluoro-l,3,5-tri-O-benzoyl- ⁇ -D-arabinofuranose as a first radiolabeled intermediate.
  • the first radiolabeled intermediate can be reacted with hydrogen bromide to form 2-deoxy-2-[ F]fluoro-3,5-di-O-benzoyl- ⁇ -D-arabinofuranosyl bromide as a second radiolabeled intermediate.
  • the second radiolabeled intermediate can be reacted with 5-(lower alkyl)-4-N-(trimethylsilyl)-2-O- (trimethylsilyl)pyrirnidine-4-amine to form 5-(lower alkyl)-l-(2'-deoxy-2'- [ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -D-arabinofuranosyl)cytosine as a third radiolabeled intermediate.
  • the third radiolabeled intermediate can be reacted with an alkoxide to form the [ 18 F]D-FRAC PET probe 5-(lower alkyl)-l-(2'-deoxy-2'-[ 18 F]fluoro- ⁇ -D- arabinofuranosyl)cytosine.
  • a lower alkyl can be an alkyl having from 1 to 6 carbons.
  • the lower alkyl can be methyl, so that the synthesized PET probe is [ 18 F]D-FMAC.
  • a method of synthesizing an [ 18 F]L-FRAC PET probe according to the present invention can include the following. 2-O-[(Trifluoromethyl)sulfonyl]- 1,3,5- tri-0-benzoyl- ⁇ -L-ribofuranose can be reacted with [ 18 F]fluoride ion to form 2- deoxy-2-[ l 8 F]fluoro-l,3,5-tri-O-benzoyl- ⁇ -L-arabinofuranose as a first radiolabeled intermediate.
  • the first radiolabeled intermediate can be reacted with hydrogen bromide to form 2-deoxy-2-[ l 8 F]fluoro-3,5-di-O-benzoyl- ⁇ -L-arabinofuranosyl bromide as a second radiolabeled intermediate.
  • the second radiolabeled intermediate can be reacted with 5-(lower alkyl)-4-N-(trimethylsilyl)-2-O- (trimethylsilyl)pyrimidine-4-amine to form 5-(lower alkyl)-l-(2'-deoxy-2'- [ 18 F]fluoro-3,5-di-O-benzoyl- ⁇ -L-arabinofuranosyl)cytosine as a third radiolabeled intermediate.
  • the third radiolabeled intermediate can be reacted with an alkoxide to form the [ 18 F]L-FRAC PET probe 5-(lower alkyl)-l-(2'-deoxy-2'-[ 18 F]fluoro- ⁇ -L- arabinofuranosyl)cytosine.
  • a lower alkyl can be an alkyl having from 1 to 6 carbons.
  • the lower alkyl can be methyl, so that the synthesized PET probe is [ 18 F]L-FMAC.
  • a method of imaging according to the invention can include the following.
  • a PET probe can be contacted with biological material.
  • PET imaging can be used to determine a local concentration of the PET probe in the biological material.
  • the local concentration of the PET probe can be correlated with a local immune response.
  • the local immune response can be the accumulation of activated T lymphocytes, and the activated T lymphocytes can take up more PET probe per cell than non-activated T lymphocytes.
  • a quantity of a PET probe for example, [ 18 F]D- FAC, can be administered to an animal or human.
  • the PET probe can be a dCK substrate and/or resistant to deamination by an enzyme, e.g., cytidine deaminase (CDA) or adenosine deaminase.
  • CDA cytidine deaminase
  • PET imaging can be used to determine a local concentration of the PET probe in the animal or human, and the local concentration of the PET probe can be correlated with a local immune response or neoplastic tissue.
  • the local concentration of the PET probe can be correlated with abnormal activity in an organ or portion of the lymphatic system, for example, in a lymph node or in the spleen.
  • the local concentration of the PET probe can be correlated with a lymphoma lesion or with a malignant lymphoid disease.
  • the animal or human can have a condition such as cancer, lymphadenopathy, melanoma, leukemia, glioma, an autoimmune disorder, a development disorder, viral infection, bacterial infection, parasitical infection, infection, a metabolic disease, inflammation, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, Experimental Autoimmune Encephalomyelitis (EAE), multiple sclerosis, and/or atherosclerosis.
  • EAE Experimental Autoimmune Encephalomyelitis
  • the PET probe can be used in the diagnosis and/or treatment of such a condition.
  • the animal or human can be undergoing a therapy such as cancer immunotherapy, immunotherapy, interferon therapy, vaccination, radiation therapy, chemotherapy, and/or antibiotic therapy.
  • the local concentration of the PET probe can be used to diagnose cancer and/or monitor cancer treatment.
  • a method of imaging according to the invention can include the following.
  • a PET probe that is a dCK substrate resistant to deamination can be contacted with a biological material.
  • the PET probe can be cytosine or adenosine analog.
  • PET imaging can be used to determine a local concentration of the PET probe in the biological material.
  • the local concentration of the PET probe can be correlated with a local immune response or neoplastic tissue.
  • the PET probe can be used to diagnose, treat, and/or monitor treatment of a condition, such as cancer, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, EAE, multiple sclerosis, and atherosclerosis.
  • the PET probe can be used to evaluate the efficacy in the treatment of cancer of an anticancer agent, e.g., cytarabine or 2'-difluorodeoxycytidine, that is taken up into cells via nucleoside transporters and deoxycytidine kinase (dCK)- mediated phosphorylation.
  • an anticancer agent e.g., cytarabine or 2'-difluorodeoxycytidine
  • a method of predicting resistance to an oncolytic prodrug according to the invention can include the following.
  • a PET probe for example, [ 18 F]D-FAC, [ 18 F]L-FAC, [ 18 F]D-FXAC, [ 18 F]L-FXAC, [ 18 F]D-FFAC, [ 18 F]L-FFAC, [ 18 F]D- FCAC, [ 18 F]L-FCAC, [ 18 F]D-FBAC, [ 18 F]L-FBAC, [ 18 F]D-FRAC, [ 18 F]L-FRAC, [ 18 F]D-FMAC, [ 18 F]L-FMAC, 18 F-CA, D-2- 18 F-CA, L-2- 18 F-CA, D-3- 18 F-CA, or L- 3- 18 F-CA, can be contacted with a neoplasm.
  • the cells in the neoplasm can be leukemia, acute non-lymphocytic leukemia, acute lymphocytic leukemia, blast phase of chronic myelocytic leukemia, meningeal leukemia, pancreatic cancer, ovarian cancer, breast cancer, non-small cell lung cancer, B-cell chronic lymphocytic leukemia, hairy cell leukemia, relapsed acute lymphoblastic leukemia, or refractory acute lymphoblastic leukemia cells.
  • the representative neoplastic cells that express dCK can be L1210 murine leukemia cells and the representative neoplastic cells that do not express dCK can be Ll 210- 1OK murine leukemia cells.
  • PET imaging can be used to determine a local concentration of the PET probe in the neoplasm.
  • the local concentration of the PET probe can be compared with a baseline level.
  • a local concentration of the PET probe substantially lower than the baseline level can be correlated with low dCK expression of the neoplasm.
  • Low dCK expression of the neoplasm can be correlated with oncolytic nucleoside analog resistance.
  • the baseline level can correspond, for example, to the mean of concentration of the PET probe in representative neoplastic cells that express dCK and concentration of the PET probe in representative neoplastic cells that do not express dCK.
  • the oncolytic prodrug can be cytosine arabinoside (Ara- C), fludarabine, cladribine, clofarabine, or gemcitabine.
  • a PET probe is a dCK substrate resistant to deamination by an enzyme, for example, cytidine deaminase (CDA) or adenosine deaminase.
  • CDA cytidine deaminase
  • the PET probe can be [ 18 F]L-FAC, [ 18 F]L-FXAC, [ 18 F]L-FBAC, [ 18 F]L-FCAC, [ 18 F]L-FFAC, [ 18 F]L-FRAC, [ 18 F]L- FMAC, [ 18 F]F-CA, D-2- 18 F-CA, L-2- 18 F-CA, D-3- 18 F-CA, and L-3- 18 F-CA.
  • Figure 1 is a diagram showing the identification of fluorinated deoxycytidine analogs retained in activated vs. naive T lymphocytes and incorporated into DNA.
  • Figure IA shows the relative intracellular retention of several deoxycytidine analogs;
  • Figure IB shows the chemical structure of the dFdC and D- FAC compounds;
  • Figure 1 C shows the retention of [ 3 HJdFdC and [ 3 H]D-FAC by the activated mouse CD8+T cells;
  • Figure ID shows the uptake of [ 3 H]D-FAC by cells;
  • Figure IE shows incorporation Of [ 3 H]D-FAC into the DNA of proliferating T cells.
  • Figure 2 A presents the chemical structures of 15 compounds according to embodiments of the invention.
  • Figures 2B, 2C, and 2D show microPET scans using [ 18 F]D-FAC.
  • Figure 3A presents the radiochemical synthesis of l-(2'-deoxy-2'- [ l 8 F]fluoroarabinofuranosyl)cytosine (herein, [ 18 F]D-FAC).
  • Figure 3B presents the radiochemical synthesis of 2-chloro-9-(2-deoxy-2-[ 18 F]fluoro- ⁇ -D-
  • Figure 4A through Figure 4J present the radiochemical synthesis of
  • 2-chloro-9-(2'-deoxy-2'-[ 18 F]fluoro- ⁇ -D-arabinofuranosyl)adenine herein, 2-[ 18 F]- CA), 3-[ 18 F]-CA, [ 18 F]L-FAC, [ 18 F]D-FMAC, [ 18 F]L-FMAC, [ 18 F]D-FBAC, [ 18 F]L-
  • FBAC [ 18 F]D-FCAC, [ 18 F]L-FCAC, [ 18 F]D-FFAC, and [ 18 F]L-FFAC.
  • Figure 5 shows the selectivity of [ F]D-FAC for lymphoid organs.
  • Figure 5A shows a digital whole-body autoradiograph (DWBA);
  • Figures 5B and 5C show microPET/CT scans;
  • Figure 5D shows the [ 18 F]D-FAC retention in thymocytes and splenocytes;
  • Figure 5E shows the proportion of [ 18 F]D-FAC retention per cell lineage.
  • Figure 6 shows the increased [ 18 F]D-FAC retention in spleen and lymph nodes at the peak of the primary anti-tumor immune response.
  • Figure 6A shows a microPET/CT image;
  • Figure 6B shows the accumulation of [ F]D-FAC in the spleen and lymph nodes;
  • Figure 6C shows the relative retention Of [ 18 F]D-FAC in
  • Figures 6D and 6E show microPET/CT images illustrating the accumulation of [ 18 F]FDG and [ 18 F]FLT probes.
  • FIG. 7 shows that [ 18 F]D-FAC microPET/CT allows visualization of increased lymphoid mass associated with systemic autoimmunity and can be used to monitor immunosuppressive therapeutic interventions.
  • Figure 8 is a diagram showing micro-PET scans performed on BDC-
  • Figure 8A shows microPET/CT images
  • Figure 8B presents [ 18 F]D-FAC accumulation measured in necroscopy tissue samples.
  • Figure 9 shows the chemical structures of some of the fluorinated undine, thymidine, and cytidine analogs discussed in this disclosure.
  • Figure 10 is a diagram showing the biodistribution of [ 18 F]D-FAC.
  • Figure 1OA shows decay-corrected mean time-activity curves in various organs of normal mice injected with [ 18 F]D-FAC determined from necroscopy tissue samples and indicative Of [ 18 F]D-FAC accumulation.
  • Figure 1OB shows [ 18 F]D-FAC biodistribution measured on necropsy tissue samples 60 min after injection.
  • Figure 11 is a cartoon showing potential metabolic pathways for [ 18 F]D-FAC.
  • Figure 12 is a shows [ 18 F]D-FAC microPET/CT imaging of human and murine malignancies.
  • Figure 12A shows the result of injecting SCID mice with Ba/F3 cells;
  • Figure 12B shows the result of injecting NOD SCID mice with retroviral stock;
  • Figure 12C shows the result of injecting C57BL/6 mice with B16 melanoma cells;
  • Figure 12D shows the result of injecting SCID mice with U87 glioma cells.
  • Figure 13 is a Western blot demonstrating the expression of deoxycytidine kinase (dCK) in the L1210 cell lines. The L1210 cell lines were probed with anti-total dCK antibody.
  • dCK deoxycytidine kinase
  • Figure 14 shows that deoxycytidine kinase (dCK) expression causes the retention of D-FAC.
  • Figure 14A shows the retention Of [ 3 H]D-FAC and [ 3 H]FLT probes in Ll 210, Ll 210- 10K, and Ll 210- 1OK with reintroduced dCK cell lines.
  • Figure 14B shows D-FAC phophorylation in the cell lines.
  • Figure 15 shows results of an in vivo study demonstrating that D-FAC can be used to predict resistance to widely used oncolytic prodrugs such as Gemcitabine and Ara-C.
  • Figure 15A shows accumulation of [ 18 F]FDG;
  • Figure 15B shows accumulation of [ F]D-FAC.
  • Figure 16 shows the biodistribution of [ 18 F]D-FAC in a healthy human volunteer via a coronal microPET scan 48 minutes after injection of the [ 18 F]D-FAC.
  • Figure 17 demonstrates that the intracellular accumulation (retention and phosphorylation) of [ 18 F]-CA and [ 18 F]L-FAC requires the expression of deoxycytidine kinase (dCK).
  • dCK deoxycytidine kinase
  • Figure 17A shows the accumulation of [ 18 F]D-FAC, [ 18 F]L-FAC, and [ 18 F]-CA probes in L1210-WT and L1210-10K cell lines;
  • Figure 17B shows the extent of phsophorylation of probes in L1210-WT and L1210-10K cell lysates.
  • Figure 18 shows the results of biodistribution studies Of [ 18 F]L-FAC and [ 18 F]-CA in C57/BL6 mice.
  • Figure 18A shows images obtained with [ 18 F]L- FAC
  • Figure 18B shows a [ 14 C]F-CA DWBA
  • Figure 18C shows images obtained with [ 18 F]L-FAC and microPET/CT
  • Figure 18D shows images obtained with [ 18 F]F- CA and microPET/CT.
  • Figure 19 illustrates that [ 18 F]L-FAC is more resistant to deamination than [ 18 F]D-FAC according to in vivo studies in mice and data using human plasma.
  • Figures 19A and 19B show chromatographs of [ 18 F]D-FAC and [ 18 F]L-FAC in plasma at 10 minutes and 45 minutes following injection into a mouse;
  • Figures 19C and 19D show chromatographs Of [ 18 F]D-FAC and [ 18 F]L-FAC in plasma at 10 minutes and 45 minutes after the probe was incubated with human plasma.
  • Figure 20 shows [ 18 F]L-FAC microPET images of lymphadenopathy in an animal model of systemic autoimmunity.
  • Figure 21 shows [ 18 F]L-FAC microPET images of immune activation during a primary T cell mediated anti-tumor immune response.
  • Figure 22 illustrates that [ 18 F]L-FAC can be used to predict gemcitabine resistance in vivo.
  • Figure 22A shows [ F]L-FAC microPET/CT scans;
  • Figure 22B shows [' 8 F]FDG microPET/CT scans.
  • Figure 23 illustrates that the intracellular accumulation of [ F]L-
  • FMAC requires the expression of deoxycytidine kinase.
  • Figure 24 A shows the results of biodistribution studies of L-FAC, D-
  • Figure 24B shows biodistribution [ 18 F]L-FMAC in necropsy samples.
  • Figure 25 illustrates that [ 18 F]L-FMAC is resistant to deamination.
  • Figure 25 A shows results obtained with the [ 18 F]L-FMAC standard
  • Figure 25B shows results for [ F]L-FMAC in plasma collected 45 minutes after injection.
  • Figure 26 show [ 18 F]L-FMAC microPET images of lymphadenopathy in an animal model of systemic autoimmunity.
  • Figure 26A shows results obtained with the wild type BL/6 mouse.
  • Figure 26B shows results obtained with the
  • Figure 27 shows [ 18 F]L-FMAC microPET images of immune activation during a primary T cell mediated anti-tumor immune response.
  • Figure 28 shows [ 18 F]L-FMAC microPET images of melanoma tumors in mice.
  • Figures 29A and 29B present [ 18 F]D-FAC PET images of lymphoma lesions in a human.
  • Figure 30 presents PET/CT scans of a 56 year old human male with chronic pancreatitis.
  • Figure 3OA shows an image obtained with the L-FAC probe;
  • Figure 3OB shows an image obtained with the FDG probe.
  • FDG 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • FDG 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • FDG 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • [ 18 F]D-FAC 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • FDG 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • the present invention also relates to novel methods of synthesizing PET probes disclosed herein.
  • the present invention relates to methods of using PET probes in the diagnosis and treatment of diseases and conditions involving inflammation, e.g., rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, Experimental Autoimmune Encephalomyelitis (EAE), multiple sclerosis, atherosclerosis and cancer.
  • treatment comprises prevention, partial alleviation, or cure of the condition or disorder.
  • this invention relates to methods of evaluating the usage efficacy of particular classes of anticancer agents in the treatment of cancer such as those that are taken up into cells via nucleoside transporters and deoxycytidine kinase (dCK)-mediated phosphorylation.
  • the present invention relates to methods of diagnosis and treatment of conditions that implicate cells with high deoxyribonucleoside salvage pathway activity, e.g., lymphocytes, bone marrow cells, and intestinal enterocytes.
  • the present invention relates to compositions incorporating the compounds disclosed herein.
  • the present invention relates to kits comprising any embodiment of the present invention.
  • PET Positron Emission Tomography
  • FDG 2-[ 18 F]fluoro-2-deoxy-D-glucose
  • probes used are 1- (2'-deoxy-2'-[ l 8 F]fluoro- ⁇ -D-arabinofuranosyl)cytosine (herein, [ 18 F]D-FAC), [ 18 F]- CA, [ 18 F]L-FAC, [ 18 F]D-FMAC, [ 18 F]L-FMAC, [ 18 F]D-FBAC, [ 18 F]L-FBAC, [ 18 F]D- FCAC, [ 18 F]L-FCAC, [ 18 F]D-FFAC and [ 18 F]L-FFAC.
  • the PET probes disclosed herein enabled lymphoid organ visualization by microPET that was sensitive to localized immune activation in mouse models of anti-tumor immunity.
  • the PET probes disclosed herein also detected early changes of a lymphoid mass in systemic autoimmunity and allowed for evaluation of immunosuppressive therapy. These data support the use of PET probes disclosed herein for immune monitoring and suggest a wide range of clinical applications, including for treatment visualization of certain types of cancer.
  • a radioactive uptake assay the results of which are shown in Figure 1.
  • Figure IA shows the retention profiles for tested nucleoside analogs in activated and quiescent (naive) T cells. These measurements were performed after incubating cells with radioactive compounds for 1 hr and performing successive washes to remove unincorporated probes. The structures and chemical formulas of tested compounds are shown in Figure 9. Full names of the abbreviations for the compounds are provided in Table 1. The largest difference in probe retention by proliferating compared to naive T cells was observed for 2',2'-difluorodeoxycytidine (dFdC) and was >20 fold (Figure IB).
  • dFdC 2',2'-difluorodeoxycytidine
  • the bromo compound 3 can be reacted with 4-N-(trimethylsilyl)-2-O-(trimethylsilyl)pyrimidine- 4-amine (4) to produce l-(2'-deoxy-2'-[ F]fluoro-3,5-di-O-benzoyl- ⁇ -D- arabinofuranosyl)cytosine (5).
  • the benzoyl-groups can be removed by reacting 5 with sodium methoxide to produce the PET probe, l-(2'-deoxy-2'-[ 18 F]fluoro- ⁇ -D- arabinofuranosyl)cytosine (6).
  • the bromo compound can be reacted with 2-chloroadenine to produce 2-chloro-9-(4-benzoyloxymethyl-3- benzoyloxy-2-deoxy-2-[ 18 F]fluoro- ⁇ -Q-arabinofuranosyl)adenine.
  • the benzoyl groups can be removed by reacting 2-chloro-9-(4-benzoyloxymethyl-3-benzoyloxy-2- deoxy-2-[ l 8 F]fluoro- ⁇ -Q-arabinofuranosyl)adenine with sodium methoxide to produce the PET probe, 2-chloro-9-(2-deoxy-2-[ 18 F]fluoro- ⁇ -D- arabinofuranosyl)adenine.
  • Figure 2B shows a naive BL6 mouse injected with [ 18 F]D-FAC 1 hr prior to imaging; the PET imaging shows accumulation in the spleen and the thymus, the latter of which was predicted based on elevated dCK expression in that tissue.
  • a BL6 mouse was injected with 100 micrograms of anti-CD3 antibody 24 hr prior to imaging such that a systemic i s immune response can be generated. After the 1 hr uptake of [ F]D-FAC, the probe accumulated in the spleen but there was less accumulation in the thymus because of antibody treatment.
  • [ 18 F]D-FRAC is similar to [ 18 F]D-FMAC
  • [ 18 F]L-FRAC is similar to [ 18 F]L- FMAC, except that instead of a methyl group substituted at the 5-position of the pyrimidine ring, an alkyl group having from 1 to 6 carbon atoms can be substituted at this position.
  • the compounds [ 18 F]D-FXAC and [ 18 F]L-FXAC can be useful for identifying activated T cells through deoxycytidine kinase-associated uptake detected by PET imaging.
  • [ 18 F]D-FXAC is similar to [ 18 F]D-FAC
  • [ 18 F]L-FXAC is similar to [ 18 F]L-FAC, except that instead of a hydrogen substituted at the 5-position of the pyrimidine ring, a halogen, for example, fluorine, chlorine, bromine, or iodine, can be substituted at this position.
  • a halogen for example, fluorine, chlorine, bromine, or iodine
  • lymphocyte activation can be non-invasively monitored by injecting a subject animal or human with a trace amount of an [ F] fluorine-labeled PET probe (e.g., such as in Figures 2-4), whereby the probe is expected to accumulate at sites of local immune activation and can be monitored at a whole body level using a PET scanner.
  • An [ 18 F] fluorine-labeled PET probe can be administered to an animal or a human for diagnostic purposes such as to determine the presence or extent of a disease or disorder (e.g., cancer, autoimmune disease, developmental disorder, viral infection, bacterial infection, parasitical infection, other infections, metabolic disease, or inflammation).
  • a disease or disorder e.g., cancer, autoimmune disease, developmental disorder, viral infection, bacterial infection, parasitical infection, other infections, metabolic disease, or inflammation.
  • the [ 18 F] fluorine-labeled PET probe can be administered to monitor the progress of cancer or other disease-based types of immunotherapy, interferon therapy, vaccination, radiation therapy, and antibiotic therapy.
  • developmental disorder includes immune deficiencies.
  • metabolic disease includes defects in macrophage function due to problems in enzyme storage.
  • the [ 18 F]fluorine-labeled PET probes presented in Figures 2, 3 and 4 can be administered to an animal for the purpose of developing a diagnostic technique, a therapy, or to develop a basic understanding of disease or disorder mechanisms.
  • PET imaging using probes (other than FDG) such as [ 18 F]D-FAC allows for visualization of the thymus and spleen in mice. Moreover, this technology is able to monitor alterations in the lymphoid mass and immune status under various experimental conditions. Current PET imaging work in
  • EAE shows the utility of using [ F]D-FAC for measuring key metabolic pathways in immune cells. While these probes are not exclusively retained in immune cell lineages, changes in probe accumulation throughout the body may be indicative of "disease states" and provide early biomarkers for treatment efficacy. Furthermore, the accumulation of [ 18 F]D-FAC in the thymus and spleen as well as the variations in [ 18 F]D-FAC retention at lymphoid organs during immune responses may reflect a critical role for the deoxyribonucleoside salvage pathway (as measured by the said probe) in T cell development and function.
  • mice deficient in the dCK-related gene thymidine kinase 1 display immunological abnormalities in histology and function.
  • Novel genetic mouse models of dCK deficiency and imaging via [ F]D-FAC PET may provide unique tools to dissect the immunological functions of the deoxyribonucleotide salvage pathway.
  • [ 18 F]D-FAC microPET can also be used to detect overt autoreactive immune activation in a BDC-2.5 mouse model that is prone to type 1 diabetes (Figure 8). In Figure 8A, the 1 mm coronal sections illustrate the pattern of [ 18 F]D-FAC probe accumulation in BDC-2.5 mice.
  • [ 18 F]D-FAC in contrast to FDG, the lack of [ 18 F]D-FAC retention in the heart provides the necessary low background that can enhance PET imaging of activated macrophages and other immune cells at coronary atherosclerotic lesions ( Figure 5).
  • [ 18 F]D-FAC may also be used to measure dysregulated nucleoside metabolism in cancer.
  • the NOD SCID mice were transplanted with wild type total bone marrow cells infected with MSCV-GFP-IRES-P185 BCR-ABL retroviral stocks and the leukemic mice were imaged 28 days following transplantation.
  • C57BL/6 was injected subcutaneously with 1 x 10 5 Bl 6 melanoma cells and imaged 7 days later.
  • SCID mice were injected subcutaneously with 1 x 10 6 U87 glioma cells and imaged 10 days later.
  • [ F]D-FAC may additionally be used to predict tumor responses to a particular class of anticancer agents, which include the widely used prodrugs cytarabine (Ara-C) and 2',2'-difluorodeoxycytidine (dFdC, Gemcitabine). Structurally, these prodrugs are closely related to FAC and require cellular uptake via nucleoside transporters and dCK-mediated phosphorylation for conversion to their active drug metabolites.
  • a PET biomarker to measure the cellular pharmacology of Ara-C and dFdC may assist with the stratification of susceptible and resistant tumors leading to a more rational clinical use of these important anticancer drugs.
  • Results presented here indicate that PET imaging with [ ' 8 F]D-FAC and the other inventive compounds offers new advantages in diagnostics and treatment monitoring of a wide range of disorders.
  • Findings from these studies provide the impetus for translational [ F]D-FAC PET imaging in a wide range of immunological disorders in patients.
  • the strategy used to identify and evaluate [ 18 F]D-FAC and its analogs is broadly applicable to the development of new PET probes with defined specificity for various biochemical pathways and/or immune cell lineages.
  • Biochemicals (Brea, CA): 3'-Fluoro-3'-deoxythymidine (3'-FLT); 2'-Fluoro-2'- deoxythymidine (2'-FLT); 1 -(2'-Deoxy-2'-fluoro- ⁇ -D-arabinofuranosyl)-5- methyluracil (D-FMAU); l-(2'-Deoxy-2'-fluoro- ⁇ -L-arabinofuranosyl)-5- methyluracil (L-FMAU); 2',3'-Dideoxy-3'-fluorocytidine (3'-FddC); (-)- ⁇ -2',3'- Dideoxy-5-fluoro-3'-thiacytidine (FTC); 5-Fluoro-2',3'-dideoxycytidine (5FddC); 2',2'-Difluorodeoxycytidine (dFdC); 5-Fluoro-2'-deoxyc
  • T lymphocytes from pmel-1 T cell receptor (TCR) transgenic mice were stimulated ex vivo using a melanoma antigen (1 micromolar hgplOO 25 _ 33 ). These cells were then cultured for radioactive uptake and kinase assays that were performed 72 hours post-stimulation.
  • 1 microCi of [ 3 H]D-FAC or [ 3 HJdFdC were added to a well containing 5x10 4 cells in a 96-well tissue culture plate and incubated for 1 hr at 37 0 C and 5% CO 2 .
  • the plate was then washed 5 times with media containing 5% fetal calf serum (FCS) by using the Millipore Vacuum Manifold (Billerica, MA); the amount of incorporated probe was measured by scintillation counting using the PerkinElmer Microbeta (Waltham, MA).
  • FCS fetal calf serum
  • mice were kept warm, under gas anesthesia (2% isoflurane) and injected intravenously with 200 microCi of various PET probes; 1 hr was allowed for uptake. Mice were then positioned using an imaging chamber and the data was obtained using Siemens Preclinical Solutions (Knoxville, TN) microPET Focus 220 and MicroCAT II CT systems. MicroPET data was acquired for 10 minutes and then reconstructed via statistical maximum a posteriori probability algorithms (MAP) into multiple frames 3 . The spatial resolution of PET is -1.5 mm with 0.4 mm voxel size. CT images provide a low dose (400 micron) resolution acquisition with 200 micron voxel size.
  • MAP statistical maximum a posteriori probability algorithms
  • MicroPET and CT images were co-registered using a previously described method and regions were drawn using the AMIDE software (Andreas Loening, http://amide.sourceforge.net/, v ⁇ .8.16) 4>5 . Quantification was performed by drawing 3D regions of interest (ROI).
  • ROI 3D regions of interest
  • mice used for systemic autoimmunity studies were purchased from the Jackson Laboratory (stock number 000482). To monitor the immunosuppressive treatment, these mice were given intraperitoneal injections of dexamethasone (DEX, 10 mg/kg) at 24 hr intervals and were scanned by microPET/CT 24 hr after the last injection. Animals were anesthetized with 2% isoflurane, injected intravenously with 200 microCi [ 18 F]D-FAC and then scanned with microPET/CT; mice were sacrificed immediately after imaging. Organs were rapidly excised, weighed, and the radioactivity was measured in a well counter.
  • DEX dexamethasone
  • mice were anesthetized with 2% isoflurane and injected intravenously with 1 mCi [ 18 F]D-FAC. After 1 hr, mice were euthanized, embedded in 3% carboxymethyl cellulose (CMC, Sigma), and frozen in 100% ethanol with dry ice for 45 min. The 50 micron sections were cut using a whole body cryostat, (PMV, Sweden); samples were exposed overnight on BAS-TR2025 plates (Fujifilm Life Science, Stamford, CT). Imaging plates were read using a Fujibas-5000 phosphorimager (16 bit, 25 micron resolution; Fujifilm Life Science). [0077] The total RNA was extracted from the purified naive CD8 T cells and
  • the absolute calls describing whether a probe set is present (P), marginally present (M), or absent (A) were generated using the Affymetrix GeneChip Operating Software vl.3 (GCOS) and expression values were calculated using the PM/MM difference model of DNA-Chip (dChip) 7 . Expression values across samples were normalized using dChip's invariant set method.
  • a gene was considered differentially expressed if the corresponding probe set fit the following criteria: absolute call was P in at least half of the samples, fold change >1.4 between baseline (naive CD8+ T cells) and experimental (activated CD8+ T cells) using the lower 90% confidence bound of fold change as defined in dChip, and expression difference between the baseline and experimental samples was >100.
  • Genes involved in the nucleoside de novo biosynthesis and salvage pathways were taken from the KEGG database (pathway IDs 00230 and 00240, respectively) and the corresponding probe sets were manually extracted from Affymetrix' s NetAffx to ensure complete coverage of all nucleoside pathway genes (239 probe sets) plus the SLC28 and SLC29 transporters (10 probe sets) 8-9 .
  • Pre-designed TaqMan assays were purchased from Applied Biosystems for dCK (Assay ID Mm00432794_ml), Slc29al (Assay ID Mm00452176_ml), and Slc28a3 (Assay ID Mm00627874_ml).
  • TaqMan beta-actin (Applied Biosystems, Part: 4352341E) reagents were used as an endogenous control for quantification.
  • the samples were ran out on a 48-well StepOne Real-Time PCR System (Applied Biosystems) and were analyzed with the StepOne Software v2.0 (Applied Biosystems) using the comparative Ct method ( ⁇ Ct).
  • the qPCR mixture (20 ⁇ L) contained 15 ng cDNA, TaqMan buffer, 5.5 mM MgCl 2 , 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 400 ⁇ M dUTP, the appropriate TaqMan assay, 0.5U AmpliTaq Gold, and 0.2U uracil-N-glycosylase (UNG).
  • Each assay included cDNA template in triplicates.
  • NOD SCID sublethally irradiated (275 rads) one day prior to reconstitution.
  • Whole bone marrow was isolated from the tibias and femurs of 4-8 week old wildtype mice and infected with MSCV-GFP-IRES-P185 BCR-ABL retroviruses. Three hours after infection, bone marrow cells were injected intravenously by the tail vein into recipient NOD SCID mice. Animals were monitored daily for signs of illness during a period of two months as previously described 10 .
  • the presentation, mention, or discussion of a chiral chemical compound also implies the presentation, mention, or discussion of each of the enantiomers of that chemical compound and their racemic mixtures.
  • the presentation, mention, or discussion of a chemical compound with a specified chirality also implies the presentation, mention, or discussion of the enantiomer of that chemical compound with specified chirality and racemic mixtures of these.
  • Example 1 Differential screening to identify potential PET probes sensitive to changes in nucleoside flux during T cell activation and proliferation.
  • An in vitro assay (Figure 1) was used to measure the retention of 3H- labeled nucleoside analogs (NA) in na ⁇ ve and proliferating primary T cells. Selection criteria for tested NA accounted for the known propensity of fluorine substitutions to significantly change the stereoelectronic and biochemical properties of nucleosides. Thus, only deoxyribonucleosides containing 'cold' fluorine ( 19 F) atom substitutions were tested (Figure 9).
  • T lymphocytes from pmel-1 T cell receptor (TCR) transgenic mice were stimulated ex vivo using a melanocyte/melanoma antigen (hgpl00 25 - 33 ); after 72 hrs, the proliferating T cells were incubated for 1 hr with 3 H-labeled (1 microCi) deoxyribonucleoside analogs (see Figure 9). Following successive washes, intracellular radioactivity was measured by scintillation counting.
  • This part of the figure shows the retention profiles for tested NA in activated and na ⁇ ve T cells and the striking differences likely reflect differential expression of nucleoside transporters and kinases sensitive to the nucleobase structure and to fluorine substitutions of native hydrogen and hydroxyl groups.
  • l-(2'- deoxy-2'-fluoro- arabinofuranosyl) cytosine is a dFdC analog, amenable to 18 F labeling.
  • D-FAC l-(2'- deoxy-2'-fluoro- arabinofuranosyl) cytosine
  • Figure 1C shows the retention of [ 3 HJdFdC and [ 3 H]D-FAC by the activated mouse CD8+ T cells; notice that the Y-ara analog l-(2'- deoxy-2'-D- fluoroarabinofuranosyl) cytosine (D-FAC) resembled dFdC biochemically as indicated by their similar retention in proliferating CD8+ T cells.
  • D-FAC Y-ara analog l-(2'- deoxy-2'-D- fluoroarabinofuranosyl) cytosine
  • Figure ID the increased uptake of [ 3 H]D-FAC was observed in NIH3T3 fibroblasts that were engineered to overexpress nucleoside kinases (dCK) and the nucleoside transporter SLC29A1. Note that [ 3 H]FLT was used as a positive control for TKl expressing cells.
  • Figure IE [ 3 H]D-FAC is incorporated in the DNA of proliferating
  • Example 2 Biochemical mechanisms of D-FAC retention in proliferating T cells.
  • Increased retention of D-FAC in proliferating T cells compared to na ⁇ ve T cells may reflect any one or combination of several biochemical events: (i) upregulation of nucleoside transporters; (ii) elevated phosphorylation by deoxyribonucleoside kinases leading to intracellular trapping of charged products; and (iii) increased incorporation into the DNA.
  • Gene expression analyses by microarray and qPCR were performed in T cells before and after activation (at 72 hrs) to determine the transcriptional status of specific nucleoside transporters and kinases.
  • dCK deoxycytidine kinase
  • TK2 thymidine kinase 2
  • [ 3 H]D-FAC retention in proliferating T cells reflects upregulation of SLC29A1 and that this allows increased availability of intracellular substrate and/or of dCK, which in turn leads to increased phosphorylation capacity.
  • SLC29A1 and dCK were both previously described to be involved in the metabolism of dFdC, a FAC-related nucleoside 12 ' 13 .
  • Example 3 [ 18 FlD-FAC has greater specificity for lymphoid organs than PET probes currently used to measure nucleoside metabolism and glycolysis.
  • Figure 11 shows the potential biochemical pathway measured by
  • D-FAC [ 18 F]D-FAC.
  • Putative transporters for D-FAC include SLC28A1, SLC28A3 and SLC29A1; however, only SLC29A1 is expressed in naive and proliferating cells.
  • D-FAC is phosphorylated by deoxycytidine kinase (dCK) and can also be converted to its uracil metabolite D-FAU (l-(2'-deoxy-2'-fluoro- ⁇ -D- arabinofuranosyl)uracil) by cytidine deaminase (CDA), which can be inhibited by 3,4,5,6-tetrahydrouridine (THU).
  • dCK deoxycytidine kinase
  • D-FAU l-(2'-deoxy-2'-fluoro- ⁇ -D- arabinofuranosyl)uracil
  • CDA cytidine deaminase
  • D-FAC-MP Monophosphorylated D-FAC
  • CMPK cytidylate kinase
  • DCTD deoxycytidylate deaminase
  • D-FAC-DP Diphosphorylated D-FAC
  • NMEl nucleoside diphosphate kinases
  • RRM ribonucleotide reductase
  • D-FAC-TP triphosphorylated D-FAC
  • DCTD cytidine triphosphate synthase
  • CPS cytidine triphosphate synthase
  • Enzyme Commission (EC) numbers for the key enzymes involved in the nucleoside salvage pathway are as follows: CDA (3.5.4.5); CMPK (2.7.4.14); CTPS (6.3.4.2); dCK (2.7.1.74); DCTD (3.5.4.12); NMEl, NME2, NME4, NME6, and NME7 ( 2.7.4.6); NT5 (3.1.3.5); POL (2.7.7.7); RPvM (1.17.4.1); and TKl (2.7.1.21). [0088] In Figure 5A, [ 18 F]D-FAC digital whole-body autoradiography
  • Table 2 shows that amongst PET probes for nucleoside metabolic pathways and glycolysis, [ 18 F]FAC shows better selectivity for thymus and spleen than conventional probes (values are %ID/g per organ normalized to %ID/g muscle). Retention of [ 18 F]FDG in the thymus could not be measured because of signal spillover from the heart. Number of mice was 3.
  • Example 4 [ 18 FID-FAC PET imaging detects localized changes in immune status during a primary anti-tumor immune response.
  • MoMSV Moloney murine sarcoma and leukemia virus complex
  • antigen-specific T cells primed by immunodominant epitopes encoded by viral gag and env genes rapidly expand in spleen and tumor draining lymph nodes (DLNs) and then traffic to the tumor lesion which is rejected over a period of 2-5 weeks 17 ' 18 .
  • mice imaged by [ 18 F]D-FAC were also scanned on consecutive days using [ 18 F]FDG and [ 18 F]FLT.
  • elevated [ 18 F]FDG accumulation was detected on day 13 post-virus challenge not only at the tumor but also at tumor DLNs and the spleen ( Figure 6D).
  • tumor lesions accumulated high amounts of [ 18 F]FDG, namely 8.2 ⁇ 4.2 percent injected dose of activity per gram of tissue (%ID/g) tumor over background, which was defined as the contralateral muscle tissue.
  • [ 18 F]D-FAC retention in the tumor was significantly lower (1.9 ⁇ 0.3 %ID/g tumor over background).
  • the preferential accumulation of [ 18 F]D-FAC in the tumor DLNs (at 13 ⁇ 2.7 %ID/g) compared to the tumor (at 6.7 ⁇ 0.3 %ID/g) suggests that [ 18 F]D- FAC is a more specific probe than [ 18 F]FDG for imaging anti-tumor immunity in the oncoretrovirus model ( Figures 6A and 6D).
  • no detectable [ 18 F]FLT accumulation was observed on day 14 at sites of immune activation that can be clearly visualized by [ 18 F]D-FAC and [ 18 F]FDG ( Figure 6D).
  • the marked difference between these nucleoside PET probes may reflect the high level of thymidine present in the rodent serum that competes with [ 18 F]FLT and/or may reflect better sensitivity of [ 18 F]D-FAC for measuring the rate of the deoxyribonucleoside salvage pathway in immune cells.
  • Example 5 Disease and treatment evaluation using [ 18 FID-FAC PET in animal models of autoimmunity.
  • [ 18 F]D-FAC enables monitoring of a systemic autoimmune disorder such as the Fas lpr syndrome, which is a well-studied animal model resembling human systemic lupus erythematosus.
  • Fas lpr syndrome which is a well-studied animal model resembling human systemic lupus erythematosus.
  • deficient apoptosis of lymphocytes carrying the Fas lpr mutation results in lymphadenopathy, arthritis, and immune complex -mediated glomerulonephrosis.
  • [ 18 F]D-FAC positive LNs were detected only in 2 out of 19 wild type mice, whereas microPET scans of Fas lp 7J mice showed the presence of enlarged LNs in 9 out of 13 mice.
  • B6-MRL- Fas lpr /J mice were treated with dexamethasone (DEX), a synthetic glucocorticoid that has pleiotropic, potent immunosuppressive effects. As shown in Figure 7, DEX treatment (2-7 days) reduced [ F]D-FAC retention in the thymus and peripheral LNs to undetectable levels.
  • DEX dexamethasone
  • BDC-2.5 T cell receptor transgenic mice injected with the [ 18 F]D-FAC probe is a well-established animal model for type I (autoimmunity-based) diabetes.
  • Figure 8A the 1 mm coronal sections illustrate the pattern of the [ 18 F]D- FAC probe accumulation in BDC-2.5 mice.
  • [ 18 F]D-FAC accumulation is measured in necropsy tissue samples from BL/6, BALB/c, NOD Ltj and BDC-2.5 mice. The data indicate that among these strains, BDC-2.5 mice accumulate the highest levels of [ 18 F]D-FAC in the spleen and lymph nodes. The accumulation Of [ 18 F]D-FAC probe in the lymphoid organs may indicate the activated status of the autoreactive T lymphocytes in the mouse.
  • Example 6 Radiochemical synthesis of l-(2'-Deoxy-2'-r 18 F1fluoro- ⁇ -D- arabinofuranosyl) cvtosine ([ 18 F]D-FAC).
  • the residue was dried by the azeotropic distillation with acetonitrile (3 X 0.5 mL); specifically, a solution of the trifiate 1 (10 mg) in 0.7 mL of acetonitrile was added to the dry residue before the reaction mixture was heated at 165 C for 15 min in a sealed vessel. The solution was then cooled to room temperature and passed through a small cartridge of silica gel, from which the product was eluted with 5 mL of ethyl acetate. Next, the ethyl acetate solution was evaporated to dryness before 0.1 mL of 30% HBr in acetic acid solution and then 0.4 mL of dichloroethane were added sequentially.
  • This new reaction mixture was heated at 80 C in a sealed vessel for 10 min and the solution was concentrated to ⁇ 50% of the initial volume, hi the following step, 0.7 mL of toluene was added and this solution was evaporated at HO 0 C to give the bromo (compound 3).
  • a freshly prepared disilyl derivative of cytosine (4, 35 mg) was dissolved in 1 mL of dichloroethane and added to the bromo compound 3.
  • the condensation reaction was carried out at 160 0 C in a sealed vessel for 30 min before the reaction mixture was cooled to room temperature and then passed through a small column of silica gel. The product was eluted off the column using 5 mL of a solution mixture of 10% methanol with 90% dichloromethane.
  • This solution was evaporated to dryness at 100 0 C and then treated with 0.5 mL of a solution of 0.5 M sodium methoxide in methanol.
  • the reaction mixture was heated at 100 0 C for 5 min in a sealed vessel and, thereafter, the basic reaction mixture was neutralized with 0.25 mL of IM HCl in water.
  • This reaction mixture was diluted to a total volume of 3 mL with a mixture of 1% ethanol with 99% 10 mM ammonium dihydrogen phosphate in water and injected into a semi-preparative HPLC column (Phenomenex Gemini C-18 column; 25 cm X 1 cm).
  • the HPLC column was eluted with a solvent mixture of 1 % ethanol with 99% 10 mM ammonium dihydrogen phosphate at a flow rate of 5.0 mL/min.
  • the effluent from the HPLC column was monitored with a 254 nm UV detector followed by a gamma radioactive detector.
  • the chemically and radiochemical ⁇ pure [ 18 F]D-FAC was eluted off the column with a retention time of ⁇ 15 min and the radiochemical yield ranged 20-30%.
  • Example 7 Radiochemical synthesis of l-(2'-deoxy-2'-[ F]fluoro- ⁇ -L- arabinofuranosvDcvtosine ([ ' 8 F]L-FAC).
  • Example 8 Radiochemical synthesis of 2-chloro-9-(2'-deoxy-2'-[ Fifluoro- ⁇ -D- arabinofuranosvDadenine ([' 8 F]CA) (or D-2- 18 F-CA) and 2-chloro-9-(3'-deoxy-3'- r' 8 F1fluoro- ⁇ -D-arabinofuranosyl)adenine (3-' 8 F-CA) (or D-3- l 8 F-CA). [0097] The trityl protected chloroadenosine derivative mixture 2 and 3
  • the organic layer was dried with Na 2 SO 4 , filtered, evaporated, and the crude product was subjected to silica gel column chromatography with 25% ethyl acetate in hexane as the eluent to separate and isolate the pure hydroxy products 2 and 3.
  • the triflates 4 and 5 were prepared from the corresponding hydroxy derivatives 2 and 3 as follows: The hydroxy compound 2 and same for compound 3 (0.1 mmol) was dissolved in 3 mL of dichloromethane under argon before addition of 4-dimethylaminopyridine (0.18 mmol) and the cooling of the solution in an ice bath at 0° C for 10 min.
  • the triflate precursor 4 or 5 (10 mg) was then dissolved in 1 mL of acetonitrile, which was added to the dried K 18 F/Kryptofix complex and this mixture reacted at 110 0 C for 25 min.
  • the reaction mixture was cooled to room temperature and passed through a small cartridge of silica gel, which was eluted with 4 X 2 mL of ethyl acetate. The ethyl acetate was evaporated to dryness and the residue was then dissolved in 0.5 mL of acetonitrile.
  • One mL of IM HCl was added to the acetonitrile solution and heated at 100 0 C for 5 min.
  • the reaction mixture was diluted to a total volume of 3 mL with a solution of 15% ethanol with 85% 25 mM ammonium acetate in water and injected into a semi-preparative HPLC column (Phenomenex Gemini C- 18 column; 25 X 1 cm).
  • the resulting mixture was eluted with a mobile phase of 15% ethanol with 85% 25 mM ammonium acetate in water at a flow rate of 5.0 mL/min.
  • the chemically and radiochemically pure l 8 F-labeled products 6 and 7 with retention times of 11-13 min were thus isolated in 10-15% radiochemical yields.
  • Example 8B Radiochemical synthesis of 2-chloro-9-(2-deoxy-2-[ Flfluoro- ⁇ -L- arabinofuranosyl)adenine (L-2- 18 F-CA) and 2-chloro-9-(3-deoxy-3-r 18 Flfluoro- ⁇ -L- arabinofuranosyDadenine (L-3- F-CA).
  • the title compounds can be synthesized via the reaction scheme shown above using L-2-chloroadenosine (the enantiomer of D-2-chloroadenosine).
  • [ F]D-FAC PET can be used to determine the reasons for drug resistance of tumors to oncolytic nucleoside analogs (NAs).
  • Oncolytic drugs such as gemcitabine (Gemzar) and Ara-C are widely used to treat a variety of hematological malignancies and solid tumors.
  • primary or acquired resistance to these NAs and other related prodrugs represent a significant problem to cancer treatment (Table 3).
  • Table 3 presents prodrug nucleoside analogs that require dCK (deoxycytidine kinase) for activation and pharmacodynamic effects. Previous studies have shown that dCK deficiency is a key determinant of resistance to gemcitabine and Ara-C 19>2 °.
  • Drug resistance of tumors has also been linked to decreased expression of nucleoside transporters (e.g., SLC29A1/ENT1) and deoxycytidine kinase (dCK); also, the upregulation of cytidine deaminase (CDA), cytidylate deaminase (DCTD), 5' nucleotidases, and ribonucleoside reductase have caused drug resistance (see review 23 ). From these mechanisms, [ 18 F]D-FAC and its analogs may be used to estimate gemcitabine and Ara-C resistance via decreased expression of nucleoside transporters and/or dCK. We focused on the latter mechanism since dCK represents the rate-limiting step of pro-drug activation 24 .
  • nucleoside transporters e.g., SLC29A1/ENT1
  • dCK deoxycytidine kinase
  • CDA cytidine deaminase
  • DCTD cyt
  • the molecular defect responsible for resistance in the L1210 1OK cells is loss of dCK expression due to a genetic modification of chromosome 5 in the 3' region of the dCK gene.
  • the 1OK cell line was derived by exposing the parental L1210 cells to increasing concentrations of gemcitabine 20 ; before experiments, we confirmed that L1210 1OK cells lack expression of dCK at the protein level ( Figure 13).
  • Figure 14B presents a [3H]D-FAC kinase assay with the Ll 210 cell lines using [3H]D-FAC as a substrate (1 microgram protein/reaction).
  • [ 18 F]D-FAC was evaluated in humans to a preliminary degree, whereby we investigated the normal biodistribution and radiation dosimetry of [ 18 F]D-FAC in human subjects (Figure 16). Biodistribution data were obtained from attenuation-corrected whole-body PET scans of 3 healthy male subjects after a bolus injection of [ 18 F]D-FAC (8.6 ⁇ 2.3 mCi). Emission scans were acquired 20, 48, and 76 min post injection and radiation dosimetry estimates were calculated using the Olinda® software. The organs with the highest accumulation of [ 18 F]D-FAC were the bladder, the kidneys, the spleen, the salivary glands, and the heart.
  • the organs receiving the highest absorbed doses were the urinary bladder wall (2.06E-01 rem/mCi), followed by the kidneys (1.06E-01 rem/mCi), spleen (7.36E-02 rem/mCi), osteogenic cells (6.69E-02 rem/mCi), the heart wall (5.92E-02 rem/mCi), and the small intestine (5.80E-02 rem/mCi) with the effective dose overall being 5.08E-02 rem/mCi.
  • the radiation dosimetry estimates show high agreement with the dosimetry results obtained from the studies in mice.
  • the dose-limiting organs were the urinary bladder wall and the kidneys.
  • the human [ 18 F]D-FAC scan shown in Figure 16 resembles certain aspects of the [ F]D-FAC images acquired in mice, namely that the probe accumulates in the spleen and to a lesser extent in the bone marrow of spinal column. However, in contrast to mice, the retention of [ F]D-FAC in the GI tract is substantially lower in humans.
  • Example 12 Development and evaluation of novel dCK 18 F-labeled substrates with improved in vivo stability and specificity
  • dCK deoxycytidine kinase
  • [ 18 F]L-FAC is a novel non-natural analog of D-FAC developed based on the differential enantioselectivity of dCK and CDA towards D- and L-nucleosides (note that while dCK phosphorylates both natural D- enantiomers and non-natural L-enantiomers, CDA has a strict requirement for D- deoxycytidine analogs).
  • [ 18 F]-CA ( Figures 2A and 4) is a purine dCK substrate resistant to deamination by adenosine deaminase. [00107] The observation that the intracellular accumulation of [ 18 F]-CA and
  • [ 18 F]L-FAC requires the expression of dCK is demonstrated in Figure 17 utilizing the L1210 wild type cells and the 1OK dCK deficient cells. Each cell type was incubated separately with each of the following: [ 18 F]L-FAC, and [ 18 F]-CA, and the positive control [ 18 F]D-FAC. The cells were incubated with [ 18 F]L-FAC, [ 18 F]-CA, and [ 18 F]D-FAC for 1 hour. Following successive washes, intracellular radioactivity was measured by scintillation counting to obtain the result shown in Figure 17A. By measuring intracellular radioactivity, it was determined that retention and phosphorylation Of [ 18 F]L-FAC and [ 18 F]-CA requires dCK expression.
  • Figure 17B shows results obtained from incubation of L1210 WT and 1OK cell lysates with 18 F]L- FAC, [ 18 F]-CA, and [ 18 F]D-FAC; phosphorylated products were measured by scintillation counting.
  • PET images show biodistribution studies of [ 18 F]L-FAC and [ 18 F]-CA in mice ( Figure 18).
  • Figure 18A presents images obtained with [ 18 F]L-FAC.
  • Figure 18B presents a [ 14 C]F-CA DWBA with corresponding tissue sections. C57/BL6 mice were scanned by microPET/CT using [ 18 F]L-FAC ( Figure 18C) and [ 18 F]F-CA ( Figure 18D).
  • mice were imaged 60 minutes after intravenous injection of probes. Images are 1 mm thick sagittal, coronal, and transverse slices. Percent ID/g is the percent injected dose per gram of tissue.
  • the labels are as follows: B, Bone Marrow/Bone; BL, Bladder; BR, Brain; GB, Gall Bladder; GI, Gastrointestinal tract; H, heart; K, kidney; L, Liver; LU, Lung; SP, Spleen; Thy, Thymus; ST, Stomach.
  • the results of Figure 18 demonstrate that the biodistribution of [ F]L-FAC resembles that of [ 18 F]D-FAC. In contrast, [ 18 F]-CA did not accumulate in thymus and spleen; the [ 18 F]L-FAC compound was thus further evaluated in C57/BL6 mice that were injected with the probes.
  • FIG. 19 The chromatograph in Figure 19 indicates that [ 18 F]L-FAC is more resistant to deamination than [ 18 F]D-FAC.
  • Figure 19 presents chromatographs of [ 18 F]D-FAC ( Figure 19A) and [ 18 F]L-FAC ( Figure 19B) in plasma at 10 minutes and 45 minutes following intravenous injection of the probe into a C57BL/6 mouse.
  • Figure 19 presents chromatographs of [ 18 F]D-FAC ( Figure 19C) and [ 18 F]L-FAC ( Figure 19D) in plasma 10 minutes and 45 minutes after the probe was incubated with human plasma.
  • Figure 20 the chromatograph of [ 18 F]L-FAC has improved stability relative to [ 18 F]D-FAC.
  • the labels are as follow: B, Bone Marrow/Bone; BL, Bladder; GI, Gastrointestinal tract; H, Heart; SP, Spleen; TU, Tumor; LN, Lymph Node.
  • Figure 22 shows that [ 18 F]L-FAC microPET/CT can be used to visualize leukemia cells that are dCK positive and thus predict gemcitabine resistance in vivo in a SCDD mouse model. That is, [ 18 F]L-FAC microPET/CT allows visualization of dCK positive, gemcitabine sensitive L1210 leukemia cells, but not visualization of the dCK negative, gemcitabine negative L1210 1OK subline.
  • FIG. 22A shows [ 18 F]L-FAC microPET/CT scans; and Figure 22B shows [ 18 F]FDG microPET/CT scans.
  • FIG 23 shows that retention and phosphorylation of [ 18 F]L-FMAC requires dCK expression. Lysates from L1210 wildtype (WT) and dCK deficient (10K) cells were incubated for 20 minutes with [ 18 F]L-FMAC; phosphorylated products were measured by scintillation counting.
  • Figure 24A shows the biodistribution of [ 18 F]L-FMAC from PET measurements;
  • Figure 24B shows the biodistribution of [ 18 F]L-FMAC from necroscopy data.
  • Figure 25 shows that [ 18 F]L- FMAC is resistant to deamination in mice.
  • Figure 25A shows results obtained with the [ 18 F]L-FMAC standard; and Figure 25B shows results for [ 18 F]L-FMAC in plasma collected 45 minutes after injecting the probe into mice.
  • Figure 26 shows that [ 18 F]L-FMAC microPET/CT allows visualization of increased lymphoid mass in systemic autoimmunity.
  • Figure 26A shows results obtained with the wild type BL/6 mouse.
  • Figure 26B shows results obtained with the B6.MRL-Fas ⁇ pr /J autoimmune mouse. Images are 60 minutes after intravenous injection of [ 18 F]L-FMAC and show three 1 mm thick coronal slices from mice.
  • Thy Thymus
  • LN Lymph Nodes
  • BM Bone- Marrow/Bone
  • Bl Bladder
  • GI Gastrointestinal tract
  • GB Gall Bladder
  • Sp Spleen
  • K Kidney
  • SC Spinal Column.
  • FIG. 27 shows PET/CT imaging of localized immune activation in a model of cancer immunotherapy.
  • Figure 28 shows the results of [ 18 F]L-FMAC microPET/CT imaging of B16 melanoma tumors. The images shown are 2 mm coronal sections from [ 18 F]L-FMAC microPET/CT scans 1 hour after probe injection. C57BL/6 mice were injected subcutaneously with IxIO 5 B16 melanoma cells and imaged 7 days later. The labels are as follow: L, Liver; SP, Spleen; GI, Gastrointestinal tract; BL, Bladder; Tu, Tumor; SC, Spinal Column; K, Kidney.
  • Figure 29 presents PET scan images of a human subject after injection of [ 18 F]D-FAC.
  • the coronal image shows high concentration of [ 18 F]D-FAC in a lymph node 294.
  • High concentration of a PET probe, such as [ 18 F]D-FAC, in an organ or portion of the lymphatic system can be correlated with abnormal activity in the organ or portion.
  • the high concentration of [ 18 F]D-FAC in the lymph node 294 in Figure 29B can be correlated with a lymphoma lesion.
  • high concentration of a PET probe, such as [ 18 F]D-FAC can be correlated with a malignant lymphoid disease.
  • the heterogeneous spleen 292 is visible.
  • Figure 30 presents PET/CT scans of a 56 year old human male with chronic pancreatitis.
  • the pancreas head 301 is visible.
  • Microscopic examination of a biopsy sample showed a predominantly lymphocytic inflammatory infiltrate with associated degenerative small ducts.
  • the clear accumulation of L-FAC (Figure 30A) in the pancreatic inflammatory lesions indicates that this novel PET probe could be a better alternative to FDG ( Figure 30B) for diagnosis and management of such disorders in humans.
  • Figure 30A The PET probe compounds discussed herein that are dCK
  • (deoxycytidine kinase) substrates can be used to predict resistance of cancerous cells to certain oncolytic prodrugs.
  • cells that are resistant exhibit subnormal expression of dCK (see Figures 14 and 15). Because of the subnormal dCK expression, these resistant cells exhibit low uptake of the PET probe compounds that are dCK (deoxycytidine kinase) substrates.
  • cancerous cells that are not resistant express dCK and, therefore, exhibit higher uptake of the PET probe compounds that are dCK (deoxycytidine kinase) substrates.
  • administration of the PET probe compounds to a subject or patient in conjunction with PET imaging can identify and locate oncolytic prodrug resistant cancer cells, facilitating the design of an appropriate course of treatment.
  • the PET probe compound [ 18 F]D-FAC discussed herein exhibits low retention in the brain and the myocardium. This is in contrast to the conventional PET probe FDG which substantially accumulates in the brain and the myocardium.
  • [ F]D-FAC is superior to FDG for the imaging of biological processes and states in and near to the brain and heart.
  • the much lower background of [ F]D-FAC in the brain and heart allows for the visualization of biological processes and states of interest.
  • autoimmune and/or inflammatory processes such as may be associated with multiple sclerosis and atherosclerosis can be imaged with the use of [ 18 F]D-FAC.

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